Theiler's murine encephalomyelitis virus (TMEV) causes a persistent infection characterized by demyelination. This disease is one of the best animal models of multiple sclerosis. Mice bearing mutations in either of two major myelin proteins are resistant to persistent infection. TMEV is present in the axon and, from there, can infect cytoplasmic channels within myelin. Axon-to-myelin spread does not occur in myelin mutants, indicating that the infection of myelin is required for viral persistence in wild-type mice. Surprisingly, spread of TMEV from neurons to oligodendrocyte does not require lysis of the axons. Currently, the mechanism by which the virus transfers from axon to myelin and the reason why infection of myelin enables persistence are unresolved. The main objective of this research plan is to define the role of myelin in persistence of TMEV and to elucidate a potential mechanism for non-lytic cytoplasmic exchange. Specific Aims: (1) To investigate the mechanism of axon-to-myelin viral spread using co-cultured neurons and oligodendrocytes. (2) To determine the genetic requirements of neuronal infection, of axon-to-myelin spread and of persistent infection. Study Design: I will establish neuron and oligodendrocyte co-cultures in microfluidic chambers that separate the neuronal cell bodies from myelinated axons, and will test axon-to-myelin spread of TMEV. Also, using these microfluidic co-cultures, I will determine if myelin infection is receptor-dependent and will examine genetic requirements and cellular pathways necessary for axon-to-myelin spread. Additionally, I will examine the importance of autophagy and determine which myelin components are necessary for persistence by infecting mice that are genetically deficient for autophagy or myelin genes. PUBLIC HEALTH RELEVANCE: Myelin is a vital component of the central nervous system and the main target in multiple sclerosis. Experiments using TMEV, one of the best animal models for the study of multiple sclerosis, have recently established that myelin is critical for viral persistence. Elucidating the mechanism of cytoplasmic spread between axons and myelin will reveal novel insights into the axon-supporting role of myelin and could improve our understanding of the pathology of multiple sclerosis.